We have established human cell lines which elaborate modulators of in vitro granulopoiesis. Colony stimulating activity (CSA) stimulates progenitor (CFU-C) growth in man and other species and may be analogous to the erythropoietic regulatory hormone, erythropoietin. CSA in cell line conditioned medium (CM) containing serum is sensitive to pronase, alpha-chymotrypsin, and to reduction and alkylation suggesting a glycoprotein containing disulfide bonds. Filtration through a Sephacryl S-200 yields two apparent activities, one of molecular weight approximately 140,000 which stimulates mouse marrow and another of 30,000 to 40,000 daltons which stimulates mouse and human marrow. Cell line CM concentrated between 500 to 1000 daltons by ultrafiltration contains a non-cytotoxic inhibitor of granulopoiesis, colony inhibiting activity (CIA). CIA has a broad species reactivity; its molecular nature and cell line specificity remain to be established. We propose to: 1) further purify cell line CSA from serum-free cell line conditioned medium utilizing dialysis, calcium phosphate gel adsorption, ion exchange, gel permeation and affinity chromatography, and isoelectric focusing; 2) radioiodinate CSA; 3) raise rabbit anti-CSA antibody; 4) further purify CIA by ultrafiltration and by hydrophobic, ion exchange, gel permeation and thin layer chromatography; 5) continue studies on the cell line specificity of CIA as determined by its effect on the growth and maturation of granulopoietic, erythroid and lymphoid colony forming cells in viscous culture and on H3Tdr incorporation into DNA of myeloid, erythroid, megakaryocytic and lymphoid cells in liquid culture. By so doing, we will further characterize the nature of two modulators of granulopoiesis and provide key probes for further studies of the control of granulocyte production.